Concern about the safety of pertussis vaccines composed of killed whole cells of Bordetella pertussis has led to decreased use of these vaccines in several countries. Thus an urgent need exists for a safer pertussis vaccine. For this reason, efforts are underway to develop pertussis vaccines which consist of purified proteins from B. pertussis. One of the antigens which is being considered for inclusion in these vaccines is a 69 kDa protein associated with the outer membrane of virulent strains of B. pertussis. Relatively large quantities of this protein will be needed to evaluate its role in the disease process. We have therefore devised a purification scheme which is easily reproduced and which can be scaled-up in order to make sufficient quantities of the 69 kDa protein for possible vaccine use. B. pertussis cells were heated at 60 degrees C for 1 h in order to extract the 69 kDa protein. This extract was centrifuged, the supernatant was applied to a column containing DEAE-Sepharose and was eluted from the column by a linear gradient of NaCl. The fractions containing the 69 kDa protein were then pooled and applied to a column containing Affi-Gel Blue. Contaminating protein was eluted with 0.5 M potassium phosphate followed by elution of the 69 kDa protein with buffer containing 4 M urea. This preparation which was homogenous as determined by SDS-polyacrylamide gel electrophoresis was determined to be free of lipooligosaccharide and pertussis toxin. This purification scheme is simple and the protein produced by this method appears to be suitable for the study of the biochemical and immunological properties of this protein as well as for the evaluation of this protein as a vaccine candidate.